The ability to respond to double-strand DNA breaks is of fundamental importance in all living cells. In eukaryotes, the DNA-dependent protein kinase (DNA-PK) plays a role in both the repair of double-strand DNA breaks and in associated signaling processes. Evidence suggests that DNA- PK exerts it signaling functions, in part, through functional interactions with RNA polymerase II and transcription factors. The goal of the proposed studies is to understand these interactions in more detail and to evaluation their biological significance. This project is divided into two parts. The first deals with the relationship between DNA-PK and the general transcription machinery. These studies use mutant cells that lack either the regulatory component of DNA-PK (the Ku protein) or the catalytic subunit (DNA-PKcs). Nuclear extracts from the mutant cells are defective in their ability to carry out multiple rounds of transcription in an in vitro assay. A series of experiments will be undertaken in order to understand the underlying mechanisms of this transcription defect. Experiments will also be performed to test whether interaction of DNA-PK with the general transcription machinery allows DNA-PK to modulate global levels of transcription in vivo in response to DNA damage. A second part of the proposal deals with the relationship between DNA-PK and promoter-specific transcriptional activator proteins. Proposed experiments are directed toward understanding which of the many candidate factors actually interact with DNA-PK in vivo. Additional experiments will involve characterization of changes in the enzymatic properties of DNA-PK in cells subjected to radiation injury. Novel regulatory mechanisms may exist that allow a signal originating at a DNA break to be amplified and propagated throughout the nucleus. DNA-PK has emerged as a key player in a number of important processes involving DNA breaks and DNA ends. Experiments in this proposal will provide definitive information about signaling interactions that occur between DNA-PK and the transcription apparatus.